The present invention relates to the field of gene and cell therapy. It relates, in particular, to combinations of enzymes which can be used for the destruction of cells, especially proliferative cells. It also relates to vectors allowing the transfer and intracellular expression of these combinations of enzymes, as well as their therapeutic use, in particular in anticancer gene therapy.
Gene therapy, which consists in introducing genetic information into an organism or a cell, has undergone an extraordinary development over the past few years. The identification of genes involved in pathologies, the development of vectors for the administration of genes, the development of control or tissue-specific expression systems, in particular, have contributed to the development of these new therapeutic approaches. Thus, during the past 5 years, numerous clinical trials of gene or cell therapy have been undertaken in Europe and in the United States, in fields such as monogenic diseases (haemophilia, cystic fibrosis), cancer, cardiovascular diseases or disorders of the nervous system.
In the field of pathologies linked to a cellular hyperproliferation (cancer, restenosis, and the like), various approaches have been developed. Some are based on the use of tumour suppressor genes (p53, Rb), others on the use of antisenses directed against oncogenes (myc, Ras), still others on immunotherapy (administration of tumour antigens or of specific immune cells, and the like). Another approach consists in introducing into the affected cells a toxic or suicide gene capable of inducing the destruction of the said cells. Such genes are, for example, genes capable of sensitizing the cells to a pharmaceutical agent. They are generally genes encoding nonmammalian and nontoxic enzymes which, when they are expressed in mammalian cells, convert a prodrug, which is initially little or nontoxic, to a highly toxic agent. Such a mechanism of activation of prodrugs is advantageous in several respects: it makes it possible to optimize the therapeutic index by adjusting the prodrug concentration or the expression of the enzyme, to interrupt the toxicity by no longer administering the prodrug and to evaluate the mortality rate. In addition, the use of these suicide genes offers the advantage of not being specific to a particular type of tumour, but of general application. Thus, strategies based on the use of tumour suppressor genes or of anti-oncogene antisenses are applicable only to tumours exhibiting a deficiency in the said suppressor gene or an overexpression of the said oncogene. Likewise, approaches based on immunotherapy should be developed on a patient by patient basis to take into account immunorestrictions and immunocompetences. On the other hand, a strategy based on the use of a suicide gene is applicable to any type of tumour, and, more generally, to practically any type of cell.
Numerous suicide genes are described in the literature, such as, for example, the genes encoding cytosine deaminase, purine nucleoside phosphorylase or thymidine kinase, such as for example the thymidine kinases of the varicella virus or herpes simplex virus type 1.
The cytosine deaminase of Escherichia coli is capable of catalysing the deamination of cytosine to uracil. The cells which express the E. coli gene are therefore capable of converting 5-fluorocytosine to 5-fluorouracil, which is a toxic metabolite (Mullen et al. 1992 Proc. Natl. Acad. USA 89 p33).
The purine nucleoside phosphorylase of Escherichia coli allows the conversion of nontoxic analogues of deoxyadenosine to very toxic adenine analogues. As the eukaryotic enzyme does not exhibit this activity, if mammalian cells express the bacterial gene, the analogues of deoxyadenine such as 6-methylpurine-2xe2x80x2-deoxyribonucleoside will be converted to a product which is toxic for these cells (Sorscher et al. 1994 Gene Therapy 1 p233).
The thymidine kinase of the varicella virus allows the monophosphorylation of 6-methoxypurine arabinoside. If mammalian cells express this viral gene, this monophosphate is produced and then metabolized by the cellular enzymes to a toxic compound (Huber et al. 1991 Proc. Natl. Acad. USA 88 p8039).
Among these genes, the gene encoding thymidine kinase (TK) is most particularly advantageous from the therapeutic point of view because, unlike other suicide genes, it generates an enzyme capable of specifically eliminating dividing cells, since the prodrug is converted to a nondiffusible product which inhibits the synthesis of DNA. The viral thymidine kinase, and especially the thymidine kinases of the varicella virus or of the herpes simplex virus type 1, have a substrate specificity different from the cellular enzyme, and it has been shown that they are the target of guanosine analogues such as acyclovir or ganciclovir (Moolten 1986 Cancer Res. 46 p5276). Thus, ganciclovir is phosphorylated to ganciclovir monophosphate only when the mammalian cells produce the HSV1-TK enzyme, then cellular kinases allow the ganciclovir monophosphate to be metabolized to the diphosphate and then to the triphosphate which causes the synthesis of DNA to stop and leads to the death of the cell (Moolten 1986 Cancer Res. 46 p5276; Mullen 1994 Pharmac. Ther. 63 p199). The same mechanism is produced with other thymidine kinases and other guanosine analogues.
Moreover, a propagated toxicity effect (xe2x80x9cby-standerxe2x80x9d effect) was observed during the use of TK. This effect manifests itself by the destruction not only of the cells which have incorporated the TK gene, but also neighbouring cells. The mechanism of this process may be explained in three ways: i) the formation of apoptotic vesicles which contain phosphorylated ganciclovir or thymidine kinase, obtained from the dead cells, and then phagocytosis of these vesicles by the neighbouring cells; ii) the passage of the prodrug metabolized by thymidine kinase by a process of metabolic cooperation of the cells containing the suicide gene towards the cells not containing it and/or iii) an immune response linked to the regression of the tumour (Marini et al. 1995 Gene Therapy 2 p655).
For persons skilled in the art, the use of the suicide gene encoding the thymidine kinase of the herpes virus is very widely documented. In particular, the first studies in vivo on rats having a glioma show regressions of tumour when the HSV1-TK gene is expressed and when 150 mg/kg doses of ganciclovir are injected (K. Culver et al. 1992 Science 256 p1550). However, these doses are highly toxic in mice (T. Osaki et al. 1994 Cancer Research 54 p5258) and therefore completely proscribed in gene therapy in man.
A number of therapeutic trials are also underway in man, in which the TK gene is delivered to the cells by means of various vectors such as especially retroviral or adenoviral vectors. In clinical trials of gene therapy in man, much smaller doses, of the order of 5 mg/kg, have to be administered and for a short duration of treatment (14 days) (E. Oldfield et al. 1995 Human Gene Therapy 6 p55). For higher doses or for more prolonged treatments, undesirable toxic side effects are indeed observed.
To overcome these disadvantages, it has been proposed to synthesize more specific or more active thymidine kinase derivatives to phosphorylate the guanosine analogues. Thus, derivatives obtained by site-directed mutagenesis have been described. However, no precise biochemical characterization on the pure enzymes has been carried out, no cellular test using these mutants has been published and no functional improvement has been reported (WO 95/30007; Black et al., 1993 Biochemistry 32 p11618). In addition, the inducible expression of an HSV1-TK gene, deleted of its first 45 codons, has been performed in eukaryotic cells, but the prodrug doses used remain comparable to those described in all the trials in the literature (B. Salomon et al. 1995 Mol. Cell. Biol. 15 p5322). Consequently, none of the variants described up until now exhibits an improved activity in relation to thymidine or towards ganciclovir.
The present invention provides an improved method of gene therapy using a suicide gene. The present invention describes, in particular, a method making it possible to improve the efficiency of phosphorylation of the guanosine analogues by thymidine kinases and thereby to improve the therapeutic potential of this treatment. The present invention provides, especially, a method for triphosphorylating nucleoside analogues such as ganciclovir or acyclovir so that the triphosphorylation of these analogues is very significantly increased at ganciclovir doses (resp. acyclovir) i) which are significantly lower; ii) or capable of causing a more pronounced xe2x80x9cby-standerxe2x80x9d effect; iii) or not leading to a cellular toxicity which might occur when the wild-type thymidine kinase is overexpressed.
This method may be applied to cancer, to cardiovascular diseases, or to any application requiring the death of certain cells such as cells infected with a virus; this virus may be a virus of the HIV (human immunodeficiency virus), CMV (cytomegalovirus) or RSV (respiratory syncytial virus) type.
The present invention is based, in particular, on the use of a combination of enzymes which make it possible to improve, in vivo, the reaction of phosphorylation of the nucleoside analogues.
A first subject of the invention therefore consists in a composition comprising:
an enzyme capable of phosphorylating a nucleoside analogue, to generate a monophosphate analogue,
an enzyme capable of phosphorylating the said monophosphate analogue, to generate a diphosphate analogue, and,
an enzyme capable of phosphorylating the said diphosphate analogue, to generate a toxic triphosphate analogue.
More particularly, the enzyme capable of phosphorylating a nucleoside analogue, to generate a monophosphate analogue is a thymidine kinase, the enzyme capable of phosphorylating the said monophosphate analogue, to generate a diphosphate analogue is a guanylate kinase and the enzyme capable of phosphorylating the said diphosphate analogue to generate a triphosphate analogue is a nucleoside diphosphate kinase. Moreover, the compositions according to the invention may comprise, not the enzyme directly, but a nucleic acid encoding the enzyme. In this regard, the subject of the invention is also a composition which can be used for the delivery and production in vivo of a combination of enzymes, comprising:
a first nucleic acid encoding an enzyme capable of phosphorylating a nucleoside analogue, to generate a monophosphate analogue,
a second nucleic acid encoding an enzyme capable of phosphorylating the said monophosphate analogue, to generate a diphosphate analogue, and,
a third nucleic acid encoding an enzyme capable of phosphorylating the said diphosphate analogue, to generate a toxic triphosphate analogue.
Advantageously, the first nucleic acid encodes a thymidine kinase, the second nucleic acid encodes a guanylate kinase and the third nucleic acid encodes a nucleoside diphosphate kinase.
The nucleoside analogue is generally a guanosine analogue, such as for example ganciclovir, acyclovir or penciclovir. Other nucleoside analogues are for example compounds of the trifluorothymidine, 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil, ara-A, 1-beta-D-arabinofuranosylthymidine (araT), 5-ethyl-2xe2x80x2-deoxyuridine, iodouridine, AZT, AIU, dideoxycytidine, AraC and bromovinyldeoxyuridine (BVDU) type. The preferred analogues are ganciclovir, acyclovir, penciclovir and BVDU, preferably ganciclovir and acyclovir. The triphosphate form is toxic in the sense that it causes, directly or indirectly, cell death.
When mammalian cells, modified to express thymidine kinase (HSV1-TK for example), are exposed to a nucleoside analogue (ganciclovir for example), they become capable of carrying out the phosphorylation of the ganciclovir to give ganciclovir monophosphate. Subsequently, cellular kinases allow this ganciclovir monophosphate to be metabolized successively to the diphosphate and then the triphosphate. The gancyclovir triphosphate thus generated then produces toxic effects by becoming incorporated into the DNA and partly inhibits the cellular alpha DNA polymerase, thereby causing DNA synthesis to stop and therefore leads to the death of the cell. In this mechanism, the step of monophosphorylation of the nucleoside analogue is considered to be the limiting step. It is for this reason that different approaches have been described in the prior art to try to improve the intrinsic properties of thymidine kinase (creation of TK mutants, research for more efficient administration and expression systems, and the like).
The present invention shows clearly that it is possible to improve the efficacy of the treatment by administering, in combination with a thymidine kinase, other enzymes involved in the phosphorylation of nucleoside analogues. The subject of the present invention is thus various combinations of enzymes which make it possible to optimize the intracellular reaction of triphosphorylation of nucleoside analogues. Another aspect of the present invention relates to vectors allowing the introduction and intracellular expression of these combinations of enzymes. They may be in particular several vectors each allowing the production of an enzyme, or of one or more vectors each allowing the production of several enzymes or of all the enzymes. The present invention also relates to a process for the triphosphorylation of nucleoside analogues in the presence of a combination of enzymes, optionally produced in situ by expression of corresponding genes, as well as a process for the destruction of proliferative cells.
The phosphorylation of the nucleoside monophosphates to the nucleoside triphosphates and then to the triphosphates has been documented in vitro. These phosphorylations are carried out in the presence i) of human erythrocyte lysate in the case of ganciclovir (Cheng et al. 1983 J. Biol. Chem. 258 p12460) or ii) of enzymatic preparations of guanylate kinase and of nucleoside diphosphate kinase of human erythrocytes, in the case of ganciclovir and of acyclovir (Miller et al. 1980 J. Biol. Chem. 255 p720; Smxc3xa9e et al. 1985 Biochem. Pharmac. 34 p1049). Although the phosphorylation of the nucleoside monophosphates to the diphosphates and then to the triphosphates has been demonstrated in mammalian cells, these conversions do not appear to be total with ganciclovir monophosphate or acyclovir monophosphate (Agbaria et al. 1994 Mol. Pharmacol. 45 p777; Caruso et al. 1995 Virology 206 p495; Salomon et al. 1995 Mol. Cell. Biol. 15 p5322).
The present application now describes compositions which make it possible to improve the therapeutic efficacy of a thymidine kinase in vivo.
The first enzyme used in the compositions and methods according to the invention, which is capable of phosphorylating a nucleoside analogue to generate a monophosphate analogue, is advantageously a nonmammalian thymidine kinase. It is preferably a thymidine kinase of viral, and in particular herpetic, origin. Among the herpetic thymidine kinases, there may be mentioned especially the herpes simplex virus type 1 thymidine kinase (HSV1-TK), the herpes simplex virus type 2 thymidine kinase (HSV2-TK), the varicella virus thymidine kinase (VZV-TK), the Epstein-Barr virus thymidine kinase (EBV-TK), or alternatively the thymidine kinase of herpetic viruses of bovine origin (Mittal et al., J. Virol 70 (1989) 2901), equine origin (Robertson et al., NAR 16 (1988) 11303), feline origin (Nunberg et al., J. Virol. 63 (1989) 3240) or simian origin (Otsuka et al., Virology 135 (1984) 316).
The sequence of the gene encoding the thymidine kinase enzyme of the herpes simplex virus type 1 has been described in the literature (see especially McKnight 1980 Nucl. Acids Res. 8 p5931). Natural variants of it exist, leading to proteins having a comparable enzymatic activity on thymidine, or ganciclovir (M. Michael et al. 1995 Biochem. Biophys. Res. Commun 209 p966). The sequence of the gene encoding the thymidine kinase enzyme of the herpes simplex virus type 2 has also been described (Swain et al., J. Virol. 46 (1983) 1045).
The thymidine kinase used in the present invention may be a native thymidine kinase (the naturally occurring form of the enzyme or one of its naturally occurring variants), or a derived form, that is to say resulting from structural modification(s) of the native form. As indicated above, various mutants or derivatives have been described in the literature. Although their intrinsic properties do not appear to be significantly modified, these molecules may be used within the framework of the present invention. They are for example mutants having a modification close to the DRH region of the site of interaction of a nucleoside (WO 95/30007). The DRH region corresponds to the aspartic, arginine and histidine residues at positions 163, 164 and 165 of TK. These three positions are highly conserved between the herpetic TKs. Various mutants have been described at positions 160-162 and 168-170 (WO 95/30007). Other artificial variants possess a modification at the ATP-binding site (FR96 01603). Moreover, other TK derivatives may be prepared according to conventional molecular biology techniques, and used in the combinations of the invention. These mutants may be prepared, for example, by mutagenesis on a nucleic acid encoding a native, preferably herpetic, thymidine kinase or one of its variants. Numerous methods which make it possible to perform the site-directed mutagenesis or the random mutagenesis are known to persons skilled in the art and there may be mentioned the PCR- or oligonucleotide-directed mutagenesis, random mutagenesis in vitro by chemical agents such as for example hydrolylamine or in vivo in mutant E. coli strains (Miller xe2x80x9cA short course in bacterial geneticsxe2x80x9d, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1992). The sequences thus mutated are then expressed in a cellular or acellular system and the expression product is tested for the presence of a thymidine kinase type activity, under the conditions described especially in the examples. Any enzyme resulting from this process, which has the capacity to phosphorylate a nucleoside analogue, to generate a monophosphate analogue, may be used in the present invention.
Preferably, a TK derived from the herpes simplex virus type 1 thymidine kinase (HSV1-TK) or a corresponding coding nucleic acid is used within the framework of the present invention. It is more particularly HSV1-TK or one of its variants, such as the naturally occurring variants or the artificial variants. Among the artificial variants, there may be mentioned more particularly the variants P155A/F161V and F161I (Biochemistry 32 (1993) p.11618), the variant A168S (Prot. Engin. 7 (1994) p.83) or the variants having a modification at the ATP-binding site, such as the variant M60I. Still more preferably, a nucleic acid encoding the herpes simplex virus type 1 thymidine kinase (HSV1-TK) is used.
The second enzyme used in the compositions and methods according to the invention, which is capable of phosphorylating a nucleoside monophosphate analogue to generate a diphosphate analogue, is advantageously a guanylate kinase. The guanylate kinase (GMPK) was purified from various organisms (man, rat, bovine, yeast). The gene encoding GMPK has also been cloned into various cell types, and especially into the yeast Saccharomyces cerevisiae, GUK1 gene (M. Konrad 1992 J. Biol. Chem. 267 p25652). From this gene, the 20 kDa GMPK enzyme has also been purified. The GMPK gene, designated gmk, has also been isolated and overexpressed in Escherichia coli (D. Gentry et al. 1993 J. Biol. Chem. 268 p14316). This enzyme is different from the S. cerevisiae enzyme in terms of cooperativity and of oligomerization, whereas the sequences exhibit strong regions of identity, 46.2% on 182 residues. The A11042 sequence, identified as encoding a factor having a haematopoietic cell growth potential activity (EP0,274,560), exhibits 51.9% identity on 180 residues with the S. cerevisiae GUK1 gene and appears to constitute the human homologue of GMPK, although no biochemical data has been published.
The third enzyme used in the compositions and methods according to the invention, which is capable of phosphorylating a nucleoside diphosphate analogue to generate a triphosphate analogue, is advantageously a nucleoside diphosphate kinase. Nucleoside diphosphate kinase (NDPK) is an enzyme with a broad substrate specificity and has been purified from a wide variety of sources (M. Inouye et al. 1991 Gene 105 p31). For various organisms (Myxococcus xanthus, Drosophila melanogaster, Dictyostelium discoideum, rat, bovine, man, E. coli and S. cerevisiae), the gene encoding NDPK has been cloned and the corresponding enzymes are highly homologous (K. Watanabe et al. 1993 Gene 29 p141). However, only the higher eukaryotic enzymes possess a xe2x80x9cleucine zipperxe2x80x9d sequence. The human genes described which encode an NDPK activity are in particular nm23-H1 and nm23-H2. It is suggested that the nm23-H2 gene encodes a bifunctional protein with two independent functions which are NDPK activity and transcription factor (E. Postel et al. 1994 J. Biol. Chem. 269 p8627). The S. cerevisiae YNK gene is not an essential gene for the yeast and encodes NDPK which is probably a tetrameric protein in which the molecular weight of the monomers is 19 kDa (A. Jong et al. 1991 Arch. Biochem. Biophys. 291 p241).
The nucleic sequences encoding the GMPK or NDPK used within the framework of the invention may be of human, animal, viral, synthetic or semisynthetic origin.
In general, the nucleic sequences of the invention may be prepared according to any technique known to persons skilled in the art. By way of illustration of these techniques, there may be mentioned especially:
chemical synthesis, using the sequences described in the literature and, for example, a nucleic acid synthesizer,
the screening of libraries by means of specific probes, especially as described in the literature, or alternatively
mixed techniques including chemical modification (extension, deletion, substitution and the like) of sequences screened from libraries.
Advantageously, the nucleic sequences used within the framework of the invention are cDNA or gDNA sequences. The cDNA sequences are intron-free sequences obtained from RNA. The gDNA sequences are chromosome regions. In eukaryotes, they comprise one or more introns. The gDNA sequences used within the framework of the invention may comprise all or part of the is introns present in the naturally occurring gene, or one or more introns artificially introduced into a cDNA in order to increase for example the efficiency of expression in mammalian cells. The nucleic acids may encode the native enzymes or variants or derivatives having an activity of the same type. These analogous nucleic acids may be obtained by conventional molecular biology techniques, which are well known to persons skilled in the art. These may be mutagenesis, site-directed or otherwise, hybridization from libraries, deletion or insertion, construction of hybrid molecules and the like. Generally, the modifications affect at least 20% of the bases of the nucleic acid. The functionality of the analogous nucleic acids is determined as described in the examples by assaying the enzymatic activity of the expression product.
A specific composition for the purposes of the invention comprises a first nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a nucleoside diphosphate kinase. In this embodiment, the nucleoside diphosphate kinase is preferably of nonhuman eukaryotic origin. Nonhuman enzyme is understood to mean an enzyme not naturally present in human cells. It may be a viral or animal enzyme, or derived from a lower eukaryotic organism (such as a yeast). It may also be a non-naturally occurring derivative of a human enzyme, exhibiting one or more structural modifications. More preferably, the NDPK used in the present invention is chosen from yeast or bovine NDPK. These compositions may, in addition, comprise a nucleic acid encoding a guanylate kinase, such as, for example, a yeast guanylate kinase.
Another specific composition for the purposes of the invention comprises a first nucleic acid encoding a thymidine kinase and a second nucleic acid encoding a nonhuman guanylate kinase. The nonhuman GMPK may be chosen from rat, bovine, yeast or bacterial GMPK, or derivatives thereof. Preferably, the GMPK is derived from a low eukaryote, especially yeast.
According to a particularly advantageous embodiment, the nucleoside diphosphate kinase used within the framework of the present invention is of eukaryotic or animal origin. Still more preferably, it is a yeast or bovine nucleoside diphosphate kinase. The applicant has, indeed, demonstrated that, surprisingly, the nucleoside diphosphate kinase from yeast, and especially from Saccharomyces cerevisiae or that from bovines, made it possible to phosphorylate nucleoside diphosphate analogues, such as ganciclovir diphosphate or acyclovir diphosphate, to nucleoside triphosphates. In addition, the results presented in the examples clearly show that these enzymes possess, on these substrates, an activity which is highly superior to the human enzyme. Thus, in the presence of 0.675 xcexcg of human enzyme, the percentage of GCV triphosphate obtained is 1.5%, whereas in the presence of 0.75 xcexcg of yeast enzyme, it is 82.9%. Likewise, in the presence of 6.75 xcexcg of human enzyme, the percentage of GCV triphosphate obtained is 24%, whereas in the presence of 1.5 xcexcg of yeast enzyme, it is 91.1% and in the presence of 5 xcexcg of bovine enzyme, it is 92%. The same results are obtained with another nucleoside analogue, acyclovir. Thus, in the presence of 6.75 xcexcg of human enzyme, the percentage of ACV triphosphate obtained is less than 0.4%, whereas in the presence of 1.5 xcexcg of yeast enzyme, it is 8%, in the presence of 15 xcexcg of yeast enzyme, it is 81% and in the presence of 5 xcexcg of bovine enzyme, it is 1.3%. These results clearly demonstrate the advantage of using, in the combinations according to the invention, a yeast or bovine nucleoside diphosphate kinase. These results also show that the first step of phosphorylation of the analogue to the monophosphate is not necessarily the limiting step in the process and that the use of a combination of enzymes according to the invention makes it possible to increase the therapeutic potential of the treatment.
Moreover, the applicant has also shown that guanylate kinase from yeast, and especially from Saccharomyces cerevisiae, also made it possible to phosphorylate analogues of nucleoside monophosphates, such as ganciclovir monophosphate or acyclovir monophosphate, to nucleoside diphosphates with a good activity. Thus, in the presence of 2.5 xcexcg of yeast enzyme, the percentage of GCV diphosphate obtained may exceed 92% and in the presence of 74 xcexcg of yeast enzyme, the percentage of ACV diphosphate obtained is 54%. Furthermore, the results presented show that yeast guanylate kinase has a GCVMP phosphorylation rate which is twice as high as the human enzyme. Likewise, its affinity for GCVMP is greater than the affinity which the human enzyme exhibits for this substrate by a factor at least equal to 2. In total, the Vmax/Km value for the yeast enzyme for GCVMP is 4.4 times higher than the value exhibited by the human enzyme. For ACVMP, the Vmax/Km value for the yeast enzyme is 7 to 9 times higher than the value exhibited by the human enzyme for this substrate.
The applicant has also demonstrated that a coupling of these two nonhuman enzymes with thymidine kinase, for example the herpes virus type I thymidine kinase, made it possible to phosphorylate nucleoside analogues such as ganciclovir or acyclovir to triphosphate derivatives, with a very high efficiency.
According to a preferred embodiment, the compositions of the invention comprise sequences encoding guanylate kinase (EC-2.7.4.8) and/or nucleoside diphosphate kinase (EC-2.7.4.6) from yeast. More preferably, they are enzymes from the yeast S. cerevisiae. These sequences are used simultaneously with a sequence (HSV1-TK) encoding the herpes simplex virus type 1 thymidine kinase (EC-2.7.1.21) so as to make it possible to triphosphorylate the nucleoside analogues such as ganciclovir or acyclovir.
As indicated above, the compositions according to the invention may comprise a combination of enzymes or of nucleic acids allowing the in vivo production of the enzymes. They are advantageously nucleic acids. This embodiment is preferred since it allows an in vivo production of higher levels of enzymes as well as a greater therapeutic effect.
According to a first embodiment, in the compositions of the invention, the nucleic acids are carried by the same expression vector. This embodiment is particularly advantageous because only one vector has to be introduced into a mammalian cell for the desired therapeutic effect to be obtained. In this embodiment, the various nucleic acids may constitute three distinct expression cassettes inside the same expression vector. Thus, the various nucleic acids may each be placed under the control of a transcriptional promoter, of a transcriptional terminator and of distinct translation signals. It is also possible to insert several nucleic acids in the form of a polycistron whose expression is directed by a single promoter and a single transcriptional terminator. This may be performed especially by the use of IRES (Internal Ribosome Entry Site) sequences positioned between the nucleic sequences. In this regard, the expression vectors of the invention may comprise a bicistronic unit directing the expression of two nucleic acids, and optionally a separate nucleic acid encoding the third enzyme. The vectors of the invention may also comprise a tricistronic unit directing the expression of the three nucleic acids. These various embodiments are illustrated in the examples.
Preferred expression vectors for the purposes of the invention are especially:
A vector comprising:
a first nucleic acid encoding a thymidine kinase, and,
a second nucleic acid encoding a nonhuman guanylate kinase. It is preferably a yeast guanylate kinase.
A vector comprising:
a first nucleic acid encoding a thymidine kinase, and,
a second nucleic acid encoding a nucleoside diphosphate kinase. It is preferably a nonhuman eukaryotic nucleoside diphosphate kinase. More preferably, it is an NDPK of bovine or yeast origin.
Advantageously, this vector comprises, in addition, a nucleic acid encoding a guanylate kinase.
The thymidine kinase used in the vectors of the invention is advantageously a thymidine kinase of viral, especially herpetic, origin. It is preferably a thymidine kinase derived from the HSV-1 or HSV-2 virus TK.
As indicated above, in the vectors according to the invention, the various nucleic acids may be placed under the control of distinct promoters, or may constitute a polycistronic unit under the control of a single promoter. In this regard, as indicated above, the enzymes may also be produced in coupled form, the various nucleic acids being coupled in order to produce a protein carrying the various enzymatic activities. In particular, a specific embodiment of the vectors according to the invention is characterized in that the nucleic acid encoding the thymidine kinase of viral origin and the nucleic acid encoding nonhuman guanylate kinase are coupled and encode a protein carrying both the TK and GUK activities. According to another variant, in the vectors of the invention, the nucleic acid encoding the thymidine kinase of viral origin and the nucleic acid encoding the nucleoside diphosphate kinase are coupled and encode a protein carrying both the TK and NDPK activities. By way of illustration, the coupling between the enzymes is carried out by means of a peptide linker, for example of structure (G4S)n.
According to another embodiment, in the compositions of the invention, the nucleic acids are carried by several expression vectors.
As indicated below, the expression vectors may be of plasmid or viral origin. As regards vectors of viral origin, they are advantageously retroviruses or adenoviruses.
Various promoters may be used within the framework of the invention. They are sequences which allow the expression of a nucleic acid in a mammalian cell. The promoter is advantageously chosen from the promoters which are functional in human cells. More preferably, it is a promoter allowing the expression of a nucleic acid sequence in an hyperproliferative cell (cancer cell, restenosis, and the like). In this regard, various promoters may be used. This may be, for example, the actual promoter of the gene considered (TK, GMPK, NDPK). This may also be regions of different origin (responsible for the expression of other proteins, or even synthetic). This may thus be any promoter or derived sequence stimulating or repressing the transcription of a gene in a specific manner or otherwise, in an inducible manner or otherwise, in a strong or weak manner. There may be mentioned especially the promoter sequences of eukaryotic or viral genes. For example, they may be promoter sequences derived from the genome of the target cell. Among the eukaryotic promoters, there may be used in particular ubiquitous promoters (promoter of the HPRT, PGK, xcex1-actin, tubulin and DHFR genes and the like), promoters of intermediate filaments (promoter of the GFAP, desmin, vimentin, neurofilament and keratin genes and the like), promoters of therapeutic genes (for example the promoter of the MDR, CFTR, Factor VIII and ApoAI genes and the like), tissue-specific promoters (promoter of the pyruvate kinase, villin, fatty acid binding intestinal protein and smooth muscle alpha-actin gene and the like), specific cell promoters of the dividing cell type, such as cancer cells or alternatively promoters corresponding to a stimulus (steroid hormone receptor, retinoic acid receptor, glucocorticoid receptor and the like) or labelled inducible. Likewise, they may be promoter sequences derived from the genome of a virus, such as for example the promoters of the adenovirus E1A and MLP genes, the CMV early promoter, or alternatively the RSV LTR promoter and the like. In addition, these promoter regions may be modified by addition of activating or regulatory sequences, or sequences allowing a tissue-specific or predominant expression.
Another subject of the invention relates to a product comprising a combination of enzymes capable of triphosphorylating a guanosine analogue, and the said guanosine analogue, for a simultaneous or separate administration or spread out over time.
The invention also relates to a composition for the in vivo production of a toxic nucleoside triphosphate analogue comprising, packaged separately or together:
a nucleoside analogue
a nucleic acid encoding a thymidine kinase
a nucleic acid encoding a guanylate kinase, and,
a nucleic acid encoding a nucleoside diphosphate kinase.
The subject of the invention is also a composition comprising a combination of enzymes involved in the phosphorylation of nucleosides, optionally generated in situ by expression of nucleic at least one of these enzymes being of nonhuman eukaryotic origin. The combination of enzymes comprises especially a TK and an NDPK; a TK and a GMPK or a TX, a GMPK and an NDPK.
The present invention also relates to a method for the destruction of proliferative cells, comprising the administration to the said cells of a combination of enzymes comprising a TK and an NDPK. Preferably, the combination comprises, in addition, a guanylate kinase. The invention also relates to a method for the destruction of proliferative cells, comprising the administration to the said cells of a combination of enzymes comprising a TK and a GMPK.
According to this method, the cells are brought into contact with a nucleoside analogue, preferably a guanosine analogue, which is converted in the cells expressing the combination of enzymes to a toxic compound.
According to the invention, enzymes may be administered to the cells by administration of nucleic acids encoding the said enzymes.
The invention also consists in the use of a nucleoside diphosphate kinase or of a nucleic acid encoding the latter, in combination with a thymidine kinase or a nucleic acid encoding a thymidine kinase, for the preparation of a pharmaceutical composition intended for the destruction of proliferative cells.
The invention also relates to a process for the triphosphorylation of a nucleoside analogue comprising the exposure of the said analogue to a combination of enzymes, at least one of them being of nonhuman eukaryotic origin.
The present invention now provides new therapeutic agents which make it possible to interfere with numerous cellular dysfunctions. With this objective in view, the nucleic acids or cassettes according to the invention may be injected as they are at the site to be treated, or incubated directly with the cells to be destroyed or treated. It has indeed been described that naked nucleic acids may penetrate into cells without a specific vector. However, the use of an administration vector, which makes it possible to improve (i) the efficiency of the cellular penetration, (ii) the cloning (iii) the extra- and intracellular stability, is preferred within the framework of the present invention. In a particularly preferred embodiment of the present invention, the nucleic sequences are incorporated into a transfer vector. The vector used may be of chemical, plasmid or viral origin.
Chemical vector is understood to cover, for the purposes of the invention, any nonviral agent capable of promoting the transfer and expression of nucleic sequences in eukaryotic cells. These chemical or biochemical, synthetic or naturally occurring vectors represent an advantageous alternative to the naturally occurring viruses, in particular for the sake of convenience, safety and also by the absence of a theoretical limit as regards the size of DNA to be transfected. These synthetic vectors have two principal functions, compact the nucleic acid to be transfected and promote its cellular attachment as well as its passage across the plasma membrane and, where appropriate, both nuclear membranes. To overcome the polyanionic nature of the nucleic acids, the nonviral vectors all possess polycationic charges. As a representative of this type of nonviral transfection techniques, currently developed for the introduction of genetic information, there may thus be mentioned those involving complexes of DNA and of DEAE-dextran (Pagano et al., J. Virol. 1 (1967)891), of DNA and of nuclear proteins (Kaneda et al., Science 243 (1989) 375), of DNA and of lipids (Felgner et al., PNAS 84 (1987) 7413), the use of liposomes (Fraley et al., J. Biol. Chem. 255 (1980) 10431), and the like.
The use of viruses as vectors for the transfer of genes appeared as a promising alternative to these physical transfection techniques. In this regard, various viruses were tested for their capacity to infect certain cellular populations. In particular, the retroviruses (RSV, EMS, MMS, and the like), the HSV virus, the adeno-associated viruses and the adenoviruses.
The nucleic acid or the vector used in the present invention may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, or transdermal administration, and the like. Preferably, the nucleic sequence or the vector is used in an injectable form. It may therefore be mixed with any pharmaceutically acceptable vehicle for an injectable formulation, especially a direct injection at the level of the site to be treated. They may be in particular isotonic sterile solutions or dry, especially freeze-dried, compositions which, upon addition depending on the case of sterilized water or of physiological saline, allow the constitution of injectable solutions. A direct injection of the nucleic acid sequence into the patient""s tumour is advantageous because it makes it possible to concentrate the therapeutic effect at the level of the affected tissues. The doses of nucleic sequences used may be adapted according to various parameters, and especially according to the vector, the mode of administration used, the relevant pathology or the desired duration of treatment.
The invention also relates to any pharmaceutical composition comprising a combination of enzymes as defined above.
It also relates to any pharmaceutical composition comprising at least one vector as defined above.
It also relates to the use of an NDPK of yeast origin or a GMPK of yeast origin for the in vivo phosphorylation of nucleoside analogues.
Because of their antiproliferative properties, the pharmaceutical compositions according to the invention are most particularly suitable for the treatment of hyperproliferative disorders, such as especially cancers and restenosis. The present invention thus provides a particularly effective method for the destruction of cells, especially hyperproliferative cells. It is thus applicable to the destruction of tumour cells or of the smooth muscle cells of the vascular wall (restenosis). It is most particularly appropriate for the treatment of cancers. By way of example, there may be mentioned colon adenocarcinomas, thyroid cancers, lung carcinomas, myeloid leukaemias, colorectal cancers, breast cancers, lung cancers, gastric cancers, oesophageal cancers, B lymphomas, ovarian cancers, bladder cancers, glioblastomas, hepatocarcinomas, bone or skin cancers, cancers of the pancreas or cancers of the kidney and of the prostate, oesophageal cancers, cancers of the larynx, head and neck cancers, HPV-positive anogenital cancers, EBV-positive cancers of the nasopharynx and the like.
It may be used in vitro or ex vivo. Ex vivo, it consists essentially in incubating the cells in the presence of a nucleic sequence (or of a vector, or cassette or directly of the derivative). In vivo, it consists in administering to the organism an active quantity of a vector (or of a cassette) according to the invention, preferably directly at the level of the site to be treated (tumour in particular), prior to, simultaneously with and/or after the injection of the prodrug considered, that is to say ganciclovir or a nucleoside analogue. In this regard, the subject of the invention is also a method for the destruction of hyperproliferative cells which comprises bringing the said cells or part of them into contact with a combination of enzymes or of nucleic sequences as defined above, in the presence of a nucleoside analogue.
The present invention will be described more fully with the aid of the examples and figures below which should be considered to be illustrative and nonlimiting.